DEVELOPMENT OF A RAPID, RELIABLE SPECIES SPECIFIC DIAGNOSTIC ASSAY FOR ERWINIA CAROTOVORA SUBSP. ATROSEPTICA
DEVELOPMENT OF A RAPID, RELIABLE SPECIES SPECIFIC DIAGNOSTIC ASSAY FOR ERWINIA CAROTOVORA SUBSP. ATROSEPTICA FJ JAFFFE, D MATTHEWS, EJA BLAKEMORE and W COOPER National Institute of Agricultural Botany, Huntingdon Road, Cambridge CS3 OLE, UK Background and objectives Erwinia carotovora subsp. atroseptica is known to cause tuber soft-rot and the systemic vascular wilt known as blackleg in potato. Erwinia carotovora subsp. atroseptica is an extremely damaging pathogen of potato resulting in millions of pounds (Sterling) of crop loss each year. Predominating in temperate climates it damages potatoes in the field and store. Tuber treatment and attempts to produce disease free seed stock on a large scale have proven unsuccessful. Several tests to detect and quantify the presence of the pathogen have been developed. Many of these, however, are time-consuming, labour-intensive and lack sensitivity. The aim of this project is to develop a rapid reliable PCR based diagnostic test for the detection of Erwinia carotovora subsp. atroseptica. The Polymerase Chain Reaction (PCR) is specific, sensitive, quantifiable, rapid, reliable, and inexpensive. Research is underway to develop a quantitative PCR assay and evaluate it in field trials. Results and conclusions Erwinia carotovora subsp. atroseptica DNA has been successfully amplified using newly developed primers. These primers are used in a two step thermal cycle in a buffer containing 0.05% (w/v) W-1 (Promega, UK). They show high specificity for Erwinia carotovora subsp. atroseptica and low levels of artifacts compared with previously published sequences[l]. Sequencing of Erwinia carotovora subsp. atroseptica specific loci amplified by PCR has allowed the design and construction of a competitive fragment for use in a quantitative PCR test. These results are being used to develop a seed health test which will be developed for commercial use. References 1. De Boer SH, Ward LJ, 1995. Phytopathology 85, pp.854-858.